Metformin Protects Against Radiation-Induced Heart Injury and Attenuates the Upregulation of Dual Oxidase Genes Following Rat’s Chest Irradiation

Radiation-induced heart toxicity is one of the serious side effects after a radiation disaster or radiotherapy for patients with chest cancers, leading to a reduction in the quality of life of the patients. Evidence has shown that infiltration of inflammatory cells plays a key role in the development of functional damages to the heart via chronic upregulation of some pro-fibrotic and pro-inflammatory cytokines. These changes are associated with continuous free radical production and increased stiffness of heart muscle. IL-4 and IL-13 are two important pro-fibrotic cytokines which contribute to the side effects of ionizing radiation exposure. Recent studies have proposed that IL-4 through upregulation of DUOX2, and IL-13 via stimulation of DUOX1 gene expression, are involved in the development of radiation late effects. In the present study, we aimed to detect changes in the expression of these pathways following irradiation of rat’s heart. Furthermore, we evaluated the possible protective effect of metformin on the development of these abnormal changes. 20 male rats were divided into 4 groups (control, radiation, metformin treated, metformin + radiation). These rats were irradiated with 15 Gy 60Co gamma rays, and sacrificed after 10 weeks for evaluation of the changes in the expression of IL4R1, IL-13R2a, DUOX1 and DUOX2. In addition, the levels of IL-4 and IL-13 cytokines, as well as infiltration of macrophages and lymphocytes were detected. Results showed an upregulation of both DUOX1 and DUOX2 pathways in the presence of metformin, while the level of IL-13 did not show any significant change. This was associated with infiltration of macrophages and lymphocytes. Also, treatment with metformin could significantly attenuate accumulation of inflammatory cells, and upregulate these pathways. Therefore, suppression of dual oxidase genes by metformin may be a contributory factor to its protective effect.

on the development of these abnormal changes. 20 male rats were divided into 4 groups (control, radiation, metformin treated, metformin + radiation). These rats were irradiated with 15 Gy 60 Co gamma rays, and sacrificed after 10 weeks for evaluation of the changes in the expression of IL4R1, IL-13R2a, DUOX1 and DUOX2. In addition, the levels of IL-4 and IL-13 cytokines, as well as infiltration of macrophages and lymphocytes were detected. Results showed an upregulation of both DUOX1 and DUOX2 pathways in the presence of metformin, while the level of IL-13 did not show any significant change. This was associated with infiltration of macrophages and lymphocytes. Also, treatment with metformin could significantly attenuate accumulation of inflammatory cells, and upregulate these pathways. Therefore, suppression of dual oxidase genes by metformin may be a contributory factor to its protective effect.
Key words: Radiation, Metformin, Heart Injury, IL-4, IL-13; DUOX1, DUOX2 adiotherapy is a non-invasive cancer treatment modality prescribed for more than half of patients with solid tumors during their treatment course. However, acute and late detrimental effects of exposure to radiation affect the deliverable intensity of radiotherapy (1). In addition, these side effects can reduce the quality of life of cancer patients. Radiation-induced heart damage is one of the serious side effects after radiotherapy of lung and breast cancers (2,3).
Several studies have shown that in addition to the beneficial effect of radiotherapy in reducing local recurrences, there is some evidence showing that overall survival is hampered by an increased risk of non-cancerous diseases such as heart disease (4).
Radiation-induced late cardiac damages such as coronary and carotid arteries diseases, ischemic heart disease etc., have been known for some decades (5). Earlier studies showed a high risk of heart diseases for women with left sided breast cancer who underwent radiotherapy (6). An increased risk of myocardial infarction (MI) for left breast cancer has been observed in comparison with the right side (7,8).
So far, several experiments have been conducted to detect the mechanisms involved in radiation-induced heart diseases. Amongst the various factors, fibrosis and inflammation play a key role (9). The long term upregulation of some cytokines such as interleukin 1 (IL-1), IL-4, IL-13, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) have pivotal roles in the development of radiationinduced fibrosis (10). IL-4 and IL-13 are two important factors that through stimulation of reactive oxygen species (ROS) production promote the production of collagen and extracellular matrix, resulting in stiffness of normal tissue (11,12). This effect causes damage to the normal function of tissues, especially in the lung, heart, and gastrointestinal organs (13).
Although, advancements in radiotherapy techniques can improve the management of side effects, several studies have proposed some radioprotective agents for ameliorating long-term consequences. IL-4 is a key cytokine involved in the late effects of radiation especially fibrosis, and is mainly released by macrophages (14). In addition to its direct role in the late effects of radiation, it can stimulate the regulation of other pro-fibrotic and pro-inflammatory cytokines such as TGF-β and IL-13 (15). These cytokines stimulate the production of free radicals for a long period which mediates chronic oxidative injury and collagen deposition in intracellular space (16). Moreover, increased level of these cytokines can promote reduction/oxidation interaction, and stimulate carcinogenesis (17). Targeting IL-4 and IL-13 is an R interesting idea for suppressing the development of fibrosis after radiotherapy (18). However, amelioration of oxidative damage by some agents such as natural antioxidants, herbal compounds, and anti-inflammatory agents have shown promising results (19,20).
Metformin is an anti-diabetic drug which has shown some antioxidant, anti-fibrotic, and radioprotective effects (21,22). Studies proposed that these effects of metformin are a result of suppression of some ROS mediator genes such as ubiquinone oxidoreductase, NADPH oxidase 4 (NOX4), and stimulation of antioxidant enzymes (23)(24)(25). In this study, we evaluated the protective effect of metformin on radiation-induced heart injury, and regulation of IL-4 and IL-13 signaling pathways.

Administration of metformin
Metformin was purchased from Tehran Chemi Company, Tehran, Iran. It was dissolved in distilled water (20mg/ml) and administered to rats (1ml per day) orally for 9 consecutive days (4 and 5 days before and after irradiation respectively). On the day of irradiation, metformin was administered 30 min before commencement. 100 mg/kg of metformin was selected as a non-toxic dose based on a previous study (26).

Irradiation of animals
Before irradiation, all rats were anesthetized with an intraperitoneal injection of ketamine and xylazine at doses of 80 and 5 mg/kg, respectively.
In two groups, rats were irradiated locally on the chest area with a single dose of 15 Gy γ-rays and a radiation field of 6×6 cm 2 . This dose of radiation was selected according to previous studies for inducing heart injury (27). The source-to-skin distance was 60 cm with a dose rate of 109 cGy/min.

Gene expression analysis
Total RNA was isolated from homogenates of auricles using TRIzol reagent (Sina gene, Iran).
Afterwards, cDNA was synthetized using the reverse transcription kit (Takara, Japan). PCR reactions were performed in a volume of 10 containing 5 l SYBR Green master mix (Takara, Japan). The expression of each gene was detected using real-time PCR with PGM1 as internal control.
Primers for each gene are shown in table 1. Real-Time PCR efficiency for all genes was determined using the slope of a linear regression which described by Pfaffl (28). 5 samples in each group were run in duplicate.

Evaluation of IL-4 and IL-3 levels
The levels of both IL-4 and IL-13 were detected using ELISA kits (Zellbio ELISA kits, Germany) based on the manufacturer's protocol.

Histopathological evaluation
After fixation, the heart tissues were

Macrophages and lymphocytes infiltration
Histopathological evaluation showed that irradiation caused a mild increase in the infiltration of inflammatory cells, including macrophages and lymphocytes. This was more obvious for lymphocytes. However, treatment with metformin completely reversed the infiltration of inflammatory cells (Figure 3).

Discussion
Radiation-induced heart damage is one of the most threatening non-cancerous diseases following

Fig. 2. The levels of IL-4 and IL-13 in rat's heart tissues following irradiation or treatment with metformin before irradiation.
Irradiation caused a significant increase in IL-4, while treatment with metformin before irradiation decreased its level. Data also did not show any significant change in the level of IL-13 (t-test, P < 0.05, a=significant compared to control; b: significant compared to radiation group; MET= metformin treatment; RAD= Radiation group; RAD+MET= radiation plus metformin treatment). exposure to irradiation. Heart injury and subsequent diseases have been detected following whole body or partial body exposure to irradiation during a radiation disaster or radiotherapy (29). Incidence of heart diseases is one of the most common reasons of increased mortality among people who were exposed to irradiation during Hiroshima and    (45). Moreover, it has been shown that metformin alleviates inflammation, apoptosis induction, and histological changes in rat's heart tissues following doxorubicin injection (46,47).
In conclusion, the present study showed that exposing rat's chest to irradiation caused a significant upregulation of proinflammation and pro-fibrotic genes including IL4Ra1, IL-13Ra2, DUOX1, and DUOX2 in heart tissues. Moreover, infiltration of macrophages and lymphocytes increased. Treatment with metformin led to potent inhibitory effects on both histopathological changes and all mentioned genes. Our results showed that IL-13 is not involved in heart injury following exposure to irradiation.